Hepatitis C virus (HCV) infects 2 percent of the world population. In most cases acute HCV infection develops into a chronic carrier state in which viral replication persists over the lifetime of the individual. Chronic HCV infection is associated with liver injury that include cirrhosis and hepatocellular carcinoma. Our studies suggest that HCV persistence and oncogenic potential are linked to disruption of cellular translational control and apoptotic programs through viral repression of the PKR protein kinase. PKR is an important mediator of the host antiviral and antiproliferative responses induced by interferon (IFN). HCV can inhibit PKR function through the actions of the viral nonstructural 5A (NS5A) protein. Regulation of PKR function by NS5A may constitute an important mechanism of viral persistence and pathogenesis by disrupting PKR-dependent control of mRNA translation, and may provide a mechanism by which HCV resists the current IFN-based antiviral therapy. We hypothesize that HCV control of PKR facilitates viral persistence and contributes to HCV pathogenesis by blocking cellular translational control programs, attenuating the cellular IFN-induced antiviral response, and disrupting host apoptotic pathways that limit cell growth and viral replication. To investigate this hypothesis we will: 1) Determine the role of NS5A in establishing HCV infection and supporting IFN- resistant virus replication; 2) Identify the amino acid residues of NS5A the mediate regulation of PKR; 3) Determine the role of host dsRNA-signaling pathways in the antiviral response to HCV, and identify how viral alteration of these processes contributes to HCV persistence; 4) Develop and characterize NS5A transgenic mouse models of HCV pathogenesis. A novel aspect our studies will be the use of an infectious molecular clone of HCV, and an HCV translation system, to assess NS5A function during HCV replication. A genetic approach will be taken to conduct a detailed structural analysis of NS5A function in yeast and mammalian cells. Our biochemical studies will focus on defining the virus/host interactions that signal and control PKR-dependent apoptotic pathways in the host cell. Finally, we will develop a small animal model to characterize HCV pathogenesis due to NS5A regulation of PKR. These studies will provide novel insights into the mechanisms of HCV persistence and pathogenesis.